Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.

The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast.

Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δmdm32 increased mitochondrial volume, and Δfzo1 decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume. KEY POINTS: • IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria • IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume • Enlarging the mitochondria may improve mitochondria-mediated terpenoid production.

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris.

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. ß-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

Enhanced production of 3,4-dihydroxybutyrate from xylose by engineered yeast via xylonate re-assimilation under alkaline condition.

To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a 'terminator catalog' by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3'-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

Optimization of 1,2,4-butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance.

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus ß-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering.

1,2,4-Butanetriol can be used to produce energetic plasticizer as well as several pharmaceutical compounds. Although Saccharomyces cerevisiae has some attractive characters such as high robustness for industrial production of useful chemicals by fermentation, 1,2,4-butanetriol production by S. cerevisiae has not been reported. 1,2,4-butanteriotl is produced by an oxidative xylose metabolic pathway completely different from the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways conventionally used for xylose assimilation in S. cerevisiae. In the present study, S. cerevisiae was engineered to produce 1,2,4-butanetriol by overexpression of xylose dehydrogenase (XylB), xylonate dehydratase (XylD), and 2-ketoacid decarboxylase. Further improvement of the recombinant strain was performed by the screening of optimal 2-ketoacid decarboxylase suitable for 1,2,4-butanetriol production and the enhancement of Fe uptake ability to improve the XylD enzymatic activity. Eventually, 1.7 g/L of 1,2,4-butanetriol was produced from 10 g/L xylose with a molar yield of 24.5%. Furthermore, 1.1 g/L of 1,2,4-butanetriol was successfully produced by direct fermentation of rice straw hydrolysate.

Sustainable production of glutathione from lignocellulose-derived sugars using engineered Saccharomyces cerevisiae.

Glutathione has diverse physiological functions, and therefore, the demand for it has increased recently. Currently, industrial mass production of glutathione is performed from D-glucose via fermentation by the budding yeast Saccharomyces cerevisiae. However, use of D-glucose often competes with demands for various other industries, leading to high production costs. To affordably produce glutathione, we aimed to produce high amounts of glutathione from D-glucose and D-xylose, which are the main constituents of lignocellulosic biomass pre-treated with acids. Genetically engineered S. cerevisiae strains that can produce high amounts of glutathione and assimilate D-xylose were constructed and cultured in media containing D-xylose. Among these recombinant strains, a S. cerevisiae GCI (XR/XDH/XK) strain over-expressing γ-glutamylcysteine synthetase, glutathione synthetase, D-xylose reductase, xylitol dehydrogenase, and xylulokinase genes successfully consumed D-xylose in the medium and produced the highest amount of glutathione. When strains were grown in media containing D-glucose and D-xylose, the GCI (XR/XDH/XK) strain showed 4.6-fold higher volumetric glutathione production (mg/L-broth), 2.2-fold higher glutathione content (%), and 2.1-fold higher cell growth (g-cell/L-broth) than the vector control strain of YPH499 (Vector). Furthermore, when recombinant S. cerevisiae strains were grown in medium containing fermentation inhibitory materials, the GCI (XR/XDH/XK) strain produced 5.8- and higher volumetric glutathione, 2.6-fold higher intracellular glutathione, and 2.9-fold higher cell growth than the vector control YPH499 (Vector) strain. The gradual sugar consumption by recombinant S. cerevisiae strains in medium containing D-glucose and D-xylose leads to high yields of glutathione. These results indicate the potential for glutathione production from lignocellulosic materials.

Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus ß-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Cell surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate.

Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with ß-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.

Metabolomics-based development of bioproduction processes toward industrial-scale production.

Microbial biomanufacturing offers a promising, environment-friendly platform for next-generation chemical production. However, its limited industrial implementation is attributed to the slow production rates of target compounds and the time-intensive engineering of high-yield strains. This review highlights how metabolomics expedites bioproduction development, as demonstrated through case studies of its integration into microbial strain engineering, culture optimization, and model construction. The Design-Build-Test-Learn (DBTL) cycle serves as a standard workflow for strain engineering. Process development, including the optimization of culture conditions and scale-up, is crucial for industrial production. In silico models facilitate the development of strains and processes. Metabolomics is a powerful driver of the DBTL framework, process development, and model construction.

Beneficial effect of optimizing the expression balance of the mevalonate pathway introduced into the mitochondria on terpenoid production in Saccharomyces cerevisiae.

Terpenoids are used in various industries, and Saccharomyces cerevisiae is a promising microorganism for terpenoid production. Introducing the mevalonate (MVA) pathway into the mitochondria of a strain with an augmented inherent cytosolic MVA pathway increased terpenoid production but also led to the accumulation of toxic pyrophosphate intermediates that negatively affected terpenoid production. We first engineered the inherent MVA pathway in the cytosol and then introduced the MVA pathway into the mitochondria using several promoter combinations, considering the toxicity of pyrophosphate intermediates. However, the highest titer, 183 mg/L, tends to be only 5% higher than that of the strain that only augmented the inherent MVA pathway (SYCM1; 174 mg/L). Next, we hypothesized that, in addition to the toxicity of pyrophosphate, other compounds in the MVA pathway could affect the squalene titer. Thus, we constructed a combinatorial strain library expressing MVA pathway enzymes in the mitochondria with various promoter combinations. The highest squalene titer (230 mg/L) was 32% higher than that of SYCM1. The promoter set revealed that mitigation of mono- and pyrophosphate compound accumulation was important for mitochondrial usage. This study demonstrated that a combinatorial strain library is useful for discovering the optimal gene expression balance in engineering yeast.

Comprehensive metabolic profiling of Geotrichum candidum and comparison with Saccharomyces cerevisiae.

Geotrichum candidum is a dimorphic yeast used in cheese processing. To our knowledge, no major metabolites have been identified to date in G. candidum except for some amino acid and fatty acid metabolites. This has limited research on the commercial use of G. candidum. In this study, we aimed to analyze temporal changes in the intra- and extra-cellular metabolites of G. candidum and Saccharomyces cerevisiae cultured in YM medium as reference. As a result of metabolite analysis, it was observed that G. candidum tends to accumulate　pentose phosphate pathway compounds, which are involved in nucleic acid synthesis, after 48 h of cultivation when compared to S. cerevisiae. In addition, G. candidum accumulated higher amounts of the antioxidant glutathione in the medium than did S. cerevisiae. In addition, G. candidum accumulated large amounts of B vitamins such as pantothenic acid and nicotinic acid in the medium. Finally, we examined the potential of G. candidum as a host for the production of useful compounds such as pantothenic acid. When cultured in medium supplemented with the pantothenic acid precursor ß-alanine, G. candidum produced 12-fold higher amounts of pantothenic acid (30 µM) than that by S. cerevisiae. This study indicates that G. candidum accumulates various useful compounds that are dissimilar to those produced by S. cerevisiae. Furthermore, G. candidum has the potential to produce useful chemicals under appropriate culture conditions.

High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.

Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.

High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system.

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine-a precursor to phenol-we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Stability Improvement of High-Pressure-Ratio Turbocharger Centrifugal Compressor by Asymmetrical Flow Control-Part II: Nonaxisymmetrical Self-Recirculation Casing Treatment.

This is part II of a two-part paper involving the development of an asymmetrical flow control method to widen the operating range of a turbocharger centrifugal compressor with high-pressure ratio. A nonaxisymmetrical self-recirculation casing treatment (SRCT) as an instance of asymmetrical flow control method is presented. Experimental and numerical methods were used to investigate the impact of nonaxisymmetrical SRCT on the surge point of the centrifugal compressor. First, the influence of the geometry of a symmetric SRCT on the compressor performance was studied by means of numerical simulation. The key parameter of the SRCT was found to be the distance from the main blade leading edge to the rear groove (Sr). Next, several arrangements of a nonaxisymmetrical SRCT were designed, based on flow analysis presented in part I. Then, a series of experiments were carried out to analyze the influence of nonaxisymmetrical SRCT on the compressor performance. Results show that the nonaxisymmetrical SRCT has a certain influence on the performance and has a larger potential for stability improvement than the traditional symmetric SRCT. For the investigated SRCT, the surge flow rate of the compressor with the nonaxisymmetrical SRCTs is about 10% lower than that of the compressor with symmetric SRCT. The largest surge margin (smallest surge flow rate) can be obtained when the phase of the largest Sr is coincident with the phase of the minimum static pressure in the vicinity of the leading edge of the splitter blades.

Stability Improvement of High-Pressure-Ratio Turbocharger Centrifugal Compressor by Asymmetric Flow Control-Part I: Non-Axisymmetrical Flow in Centrifugal Compressor.

This is Part I of a two-part paper documenting the development of a novel asymmetric flow control method to improve the stability of a high-pressure-ratio turbocharger centrifugal compressor. Part I focuses on the nonaxisymmetrical flow in a centrifugal compressor induced by the nonaxisymmetrical geometry of the volute while Part II describes the development of an asymmetric flow control method to avoid the stall on the basis of the characteristic of nonaxisymmetrical flow. To understand the asymmetries, experimental measurements and corresponding numerical simulation were carried out. The static pressure was measured by probes at different circumferential and stream-wise positions to gain insights about the asymmetries. The experimental results show that there is an evident nonaxisymmetrical flow pattern throughout the compressor due to the asymmetric geometry of the overhung volute. The static pressure field in the diffuser is distorted at approximately 90 deg in the rotational direction of the volute tongue throughout the diffuser. The magnitude of this distortion slightly varies with the rotational speed. The magnitude of the static pressure distortion in the impeller is a function of the rotational speed. There is a significant phase shift between the static pressure distributions at the leading edge of the splitter blades and the impeller outlet. The numerical steady state simulation neglects the aforementioned unsteady effects found in the experiments and cannot predict the phase shift, however, a detailed asymmetric flow field structure is obviously obtained.

Construction of an l-Tyrosine Chassis in Pichia pastoris Enhances Aromatic Secondary Metabolite Production from Glycerol.

Bioactive plant-based secondary metabolites such as stilbenoids, flavonoids, and benzylisoquinoline alkaloids (BIAs) are produced from l-tyrosine (l-Tyr) and have a wide variety of commercial applications. Therefore, building a microorganism with high l-Tyr productivity (l-Tyr chassis) is of immense value for large-scale production of various aromatic compounds. The aim of this study was to develop an l-Tyr chassis in the nonconventional yeast Pichia pastoris (Komagataella phaffii) to produce various aromatic secondary metabolites (resveratrol, naringenin, norcoclaurine, and reticuline). Overexpression of feedback-inhibition insensitive variants of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (ARO4K229L) and chorismate mutase (ARO7G141S) enhanced l-Tyr titer from glycerol in P. pastoris. These engineered P. pastoris strains increased the titer of resveratrol, naringenin, and norcoclaurine by 258, 244, and 3400%, respectively, after expressing the corresponding heterologous pathways. The titer of resveratrol and naringenin further increased by 305 and 249%, resulting in yields of 1825 and 1067 mg/L, respectively, in fed-batch fermentation, which is the highest titer from glycerol reported to date. Furthermore, the resveratrol-producing strain accumulated intermediates in the shikimate pathway. l-Tyr-derived aromatic compounds were produced using crude glycerol byproducts from biodiesel fuel (BDF) production. Constructing an l-Tyr chassis is a promising strategy to increase the titer of various aromatic secondary metabolites and P. pastoris is an attractive host for high-yield production of l-Tyr-derived aromatic compounds from glycerol.

An ion-pair free LC-MS/MS method for quantitative metabolite profiling of microbial bioproduction systems.

Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.